Although it is generally accepted that insulin action is initiated by binding to target cell surface receptor components, little is known about events which immediately follow the insulin-receptor interactions and which lead to the hormonal response. Recent interest has centered on the possibility that receptor dynamics in the membrane and/or internalization may be important features in insulin action, but nothing is known about actual chemical changes in the receptor subsequent to hormone binding. Recent methodology developed in this laboratory allows the specific convalent labelling of a high-affinity insulin receptor in intact cells or fat and liver plasma membranes by 125I-insulin using crosslinking agents we have synthesized. This ability to insert a label on the receptor allows the receptor complex to be readily monitored on SDS gels. It exists as a disulfide linked complex (300,000 daltons) which is reduced to a 125,000 monomer by dithiothreitol. Development of a selective precipitation procedure to rapidly and specifically resolve the detergent-solubilized insulin-receptor complex from other membrane proteins using anti-insulin antibody is proposed. This should allow the determination of whether the receptor undergoes covalent modulation (e.g., incorporation of 32PO4) in intact cells and whether such modulation is controlled by other hormones or insulin itself. Since we can follow the size of the labelled receptor on acrylamide gels by autoradiography, time- and temperature-dependent proteolysis or convalent associations with other membrane components can be monitored by looking for variations in gel migration rates of the 125I-insulin-receptor obtained from fat cells incubated at various times at 37 degrees Centigrade. Crosslinkers will also be utilized to monitor whether time-dependent non-covalent associations of 125I-insulin-receptor with membrane components occurs by attempting to link such components to the 125I-insulin-receptor complex in the intact membrane followed by gel electrophoresis and autoradiography.